Rotaviruses (RVs), members of the Reoviridae family, have genomes consisting of eleven segments of double-stranded (ds) RNA. In the infectious RV particle, the genome is contained within a non-enveloped icosahedral capsid composed of three concentric protein layers. The innermost protein layer is a smooth, thin, pseudo T=1 assembly formed from 12 decamers of the core lattice protein VP2. Tethered to the underside of VP2 layer are complexes comprised of the viral RNA-dependent RNA polymerase (RdRP), VP1, and the RNA-capping enzyme, VP3. Together, VP1, VP2, VP3, and the dsRNA genome form the core of the virion. The core proteins function together to transcribe the segmented dsRNA genome, producing eleven capped plus-sense (+)RNAs. The viral RdRP uses the (+)RNAs as templates for the synthesis of the dsRNA genome. Although the RdRP alone can recognize viral (+)RNAs, the polymerase is only active when VP2 is present. The VP2-dependent activity of VP1 provides a means by which genome replication (dsRNA synthesis) can be linked with genome packaging (core assembly). Newly made (+)RNAs pass directly from the RdRP to VP3, an enzyme which introduces m7G caps at the 5'-end of the transcripts through associated guanylyltransferase and methyltransferase activities. Genome replication and core assembly take place in cytoplasmic inclusions bodies of infected cells;these structures are commonly referred to as viroplasms. Two viral nonstructural proteins, the octamer NSP2 and the phosphoprotein NSP5, direct the formation of viroplasms. The interactions of NSP2 and NSP5 with VP1, VP2, and VP3 coordinate genome replication and core assembly. &#8232;&#8232; The overriding goal of this project is to characterize the structure and function of the core proteins VP1, VP2, and VP3 and the viroplasm building-blocks NSP2 and NSP5. This includes defining the structural interfaces between the proteins and establishing how these interactions affect and regulate the activities of the proteins. Progress toward this goal in 2010-11 is summarized below. &#8232; (1) RNA recognition by the RV RdRP (VP1). Earlier studies established that VP1 binds to the conserved 3'end of RV +RNAs via sequence-dependent and sequence-independent interactions. Sequence-dependent interactions permit recognition of viral +RNAs and specify an auto-inhibited positioning of the template within the catalytic site. To analyze the importance of VP1 residues that interact with +RNAs on genome replication, we engineered mutant VP1 proteins and assayed their capacity to synthesize dsRNA in vitro. Our results showed that, individually, mutation of residues that interact specifically with RNA bases do not diminish replication levels. However, simultaneous mutations lead to significantly lower levels of dsRNA synthesis, presumably due to impaired recruitment of +RNA templates. In contrast, mutation of residues making sequence-independent contacts with RNA severely diminished replication, likely as a result of improper positioning of templates at the catalytic site. A noteworthy exception was a K419A mutation that significantly enhanced the activity of VP1. The specific chemistry of Lys419 and its position at a narrow region of the template entry tunnel suggests this residue moderates replication. Together, our findings suggest that distinct classes of VP1 residues interact with +RNA to mediate template recognition and dsRNA synthesis, yet function in concert to promote viral RNA replication at appropriate times and rates. Ogden et al (2011) Residues of the rotavirus RNA-dependent RNA polymerase template entry tunnel that mediate RNA recognition and genome replication. J Virol 85:1958-69. (2) Expression and purification of the RV RNA capping enzyme (VP3). The RNA capping enzyme VP3 is the only structural protein of RV whose atomic structure has yet to be determined. The lack of such information confounds our understanding of the capping process for RV transcripts, a complex sequence of events that requires VP3 to possess up to four enzymatic activities: an RNA triphosphatase, a guanylyltransferase, and two methyltransferases. In the past year, we have continued efforts to express and purify sufficient levels of recombinant VP3 necessary for protein crystallization. (3) Domains of the core shell protein (VP2) required for VP1 polymerase activity. Besides anchoring VP1 inside the core, VP2 serves as a cofactor that triggers the catalytic activity of VP1. This activation event leads VP1 to synthesize dsRNA genome segments. The mechanism by which the RV core shell protein activates the viral polymerase remains very poorly understood. In the past year, we completed studies aimed at defining VP2 regions critical for VP1-mediated in vitro dsRNA synthesis. By comparing the functions of proteins from several different RVs, we found that polymerase activation by the core shell protein is specific. Through truncation and chimera mutagenesis, we demonstrated that the amino termini of VP2 molecules form long tethers that cradle VP1 in the core. Although the tether interactions are important to RNA synthesis, they play a nonspecific role in VP1 activation. Our results indicate that those residues of VP2 that are responsible for polymerase-activation specificity are located on the inner face of the core shell and are distinct from the amino-terminal tethers of VP2. Based on these findings, we predict that several regions of VP2 engage the polymerase during the concerted processes of RV core assembly and genome replication. McDonald SM, Patton JT (2011) Rotavirus VP2 core shell regions critical for viral polymerase activation. J Virol 85:3095-105. (4) Role of the phosphoprotein NSP5. NSP5 plays critical roles in the formation of the viroplasm and in the assembly of progeny cores and double-layered particles within these structures. The activities of NSP5 include interaction with the other viroplasm-building block NSP2 and one or more of the core structural proteins (VP1, VP2, VP3). These activities are likely modulated through phosphorylation of specific threonine/serine residues of NSP5 by host kinases. To provide greater insight into the function of NSP5 in the viral life cycle, we have created cell lines that constitutively express small interfering RNAs (siRNAs) that target the NSP5 mRNA for degradation. Because of the lack of NSP5 expression, RV infection of these cell lines leads to little or no virus replication unless complemented with siRNA-resistant NSP5 mRNA. We are developing this system as a tool for studying the importance of NSP5 in RV replication, particularly the role that phosphorylation has on the role of NSP5 in viroplasm formation and particle assembly.